Recombinant DNA technology has now made it possible to use viruses to introduce virtually any gene of interest into almost any cell of interest. Because such viruses are engineered to “express” the gene of interest, i.e., produce the protein encoded by the gene, they are called “viral expression vectors”.
Recent attention has focused on alphaviruses, which are positive-strand RNA viruses (viruses whose nucleic acid is in the form of RNA, rather than DNA) that are transmitted to mammals via arthropods (reviewed in (16) and (17)). Positive strand RNA viruses, and in particular, alphaviruses, are especially attractive viral expression vectors for several reasons: 1) their genomes are easily manipulated in cDNA form and are infectious as naked RNA, 2) their replication cycle is exclusively cytoplasmic, 3) foreign gene expression is driven by a strong viral promoter, and 4) they have a broad host-range in vitro.
Structurally, as set forth in FIG. 1, the alphavirus genome is a single-stranded RNA, approximately 11.7 kilobases (kb) long, that is “capped” at the 5′ end and “polyadenylated” at the 3′ end. The two-thirds of the genome at the 5′ end encodes nonstructural proteins (nsPs), and the one third at the 3′ end encodes structural proteins (sPs). FIG. 1 also shows that the nonstructural proteins are responsible for both replication (copying) of the entire RNA sequence as well as transcription of a “subgenomic” RNA that leads to translation of the structural proteins (for review see (32) and (34)).
For replication, the nsPs are translated directly from the infecting viral genome, designated (+) RNA, as set forth in step 1 of FIG. 1 (steps are indicated by dark circles that contain numbers). The translation of the nsPs yields four proteins that form a “replication/transcription complex”, comprising a “replicase” and a “transcriptase.” The replicase/transcriptase mediates the synthesis of a genome-length complementary strand, designated (−) RNA, which is also termed the “antigenome”, as set forth in step 2. In step 3, the replicase/transcriptase then creates an additional copy of (+) full-length RNA using the antigenome as a template.
The antigenome also serves as the template for transcription of the last third of the genome into subgenomic mRNA, as indicated in the step prior to step 4. As noted above, the 3′ one-third of the genome encodes sPs and, accordingly, the subgenomic mRNA encodes the sPs. The sPs are encoded in the form of a large “polyprotein” that is then processed to yield a capsid protein and two envelope proteins, which are designated E1 and E2. The transcription of the subgenomic segment is mediated by nucleotides that span the junction between the nsP coding region and the sP coding region, and serve as a “promoter”. Transcription from the subgenomic promoter can yield levels of subgenomic mRNA that can reach 106 copies/cell, resulting in 108 viral structural proteins per infected cell (30).
Once the envelope proteins and capsid proteins are synthesized, as per step 4, the capsid protein interacts with the replicated genome RNA to form a “nucleocapsid”, which is then packaged by the envelope proteins. “Packaging signals” that are located within the nsP coding sequence of the genomic RNA serve to facilitate this process. Because the subgenomic RNA lacks these packaging signals, only the genomic RNA is encapsidated.
Viruses in the Togaviridae and Flaviviridae have similar enveloped, icosahedral nucleocapsid structures, and are believed to have evolved from a common ancestral virus (54). Alphaviruses and rubiviruses (eg. rubella virus), both members of the Togaviridae family, have similar genomic structures and replication cycles (see FIG. 22). The replicase/transcriptase complex is translated directly from the 5′-end of the genome, and the sPs are transcribed downstream from a subgenomic promoter present on the antisense RNA. Viruses in the Flaviviridae (eg. Dengue virus, hepatitis C virus, tick-borne encephalitis virus), all have common genome organizations and replication strategies. Unlike the togaviruses, flavivirus genomes serve as the mRNA for a polyprotein that encodes both the sPs and the nSPs. The expression of these gene products is regulated at post-translational steps; there is no subgenomic transcription in the Flaviviridae. Furthermore, the gene arrangement is inverted, i.e., the sP genes are located upstream of the nSPs. Although these viruses differ in their genomic arrangement and replication strategies, they can be substituted for the viruses described herein. For example, they can be engineered into replicon expression vectors by removing the sPs coding region ((55), (56)), and packaged into virus-like particles by providing the sPs in trans (57), using techniques substantially similar to those described herein.
Other positive strand RNA viruses that have been engineered as either live and/or replication-defective expression vectors include poliovirus (58) and coronavirus (59). Although they also differ from the alphaviruses, replicon vectors derived from these viruses may be packaged using techniques similar to those described herein.
Understanding the replication/transcription processes of the alphaviruses, as well as their nucleic acid sequence, has permitted their use as expression vectors. Several alphaviruses have been sequenced, and infectious cDNA clones have also been engineered for Sindbis virus (SV; (31)), Semliki Forest virus (SFV; (20)), Venezuelan equine encephalitis virus (VEE; (11)), and Ross River virus (RRV; (18)). Vectors based on SV, SFV and VEE have shown promise as effective gene expression systems (for reviews see (14), (21), (15)).
There are, in general, two types of alphavirus expression vectors. In one type of vector, the “replication-competent” vector, a second subgenomic promoter is added to direct the expression of a foreign (heterologous) gene. This type of double-subgenomic promoter vector expresses the foreign gene of interest, as well as all the structural components needed for viral packaging; thus, these vectors are self-replicating and self-packaging. The apparent disadvantage of such a system is the production of viable virus.
To minimize the potential production of a viable virus, the alphavirus expression vectors have been further engineered to be “replication-defective.” These vectors are created by removing the genes that encode for sPs, and substituting one or more foreign genes under the control of the subgenomic promoter. Since the nsP coding sequence remains intact, these vectors can form the replication complex and self-replicate and express the foreign gene(s). They are not self-packaging, however, because they lack the sPs which encode the capsid and envelope proteins. To package these vectors into infectious particles, the vectors can be complemented “in trans” with “helper” vectors, i.e., vectors that bear the sPs on a separate RNA molecule. For example, these vectors may be packaged by cotransfecting the vector with in vitro transcribed defective-helper (DH) RNAs that encode the viral capsid and glycoproteins (19), (5) or, alternatively, by transfecting the replicon RNA into a continuous packaging cell line which expresses DH RNAs under the regulation of a nuclear promoter (26). With either system, the helper RNA is either not packaged, or packaged with very low efficiency, since it lacks the packaging signal present within the nsP coding region.
Recombination frequently occurs, however, between alphavirus replication intermediates (including the replicon and DH RNAs) and can result in the creation of self-replication and self-packaging virus (35), (29), (37). This poses potential biosafety and regulatory concerns about the use of these packaging systems. To address these concerns, scientists have developed “split-helper” packaging systems which significantly decrease the probability of generating vectors that are able to replicate and self-package (14), (27), (33). The “split-helper” system uses two separate DH RNAs, one encoding the capsid protein and another encoding the viral glycoproteins (E2/E1). This is a costly and inefficient system, however, since the two separate DH RNAs must first be transcribed in vitro, purified, and subsequently inserted into a packaging cell that has been prepared for transfection. Numerous manipulations of the RNA and cells result in inconsistent production of replicon particles.
Cells infected with an alphavirus typically produce 103–104 infectious virus particles/cell. The production of replicon particles, by contrast, is much less efficient. Cells transfected with these vectors typically produce an average of 1–50 replicon particles per cell. The low yield of replicon particles is the result of the cumulative effects of poor in vitro transcription and cellular transfection. For example, successful expression of RNA that has been transcribed in vitro requires that the RNA be capped at the 5′ end. For the split-helper systems, which contain two separate DH RNAs, there are three RNA segments that must be capped: both helper RNAs and the replicon itself. If the efficiency of the capping of the replicon in vitro is, for example, 65% and of each DH RNA is 85%, then the efficiency of the transfection is at best 42% (0.65×0.8×0.8). Thus, the efficiency of expression is limited by the efficiency of the three capping reactions and the transfection process.
Compounding the capping problem is the fact that transfection procedures using chemical reagents are relatively ineffective. Electroporation methods, where RNAs are introduced into cells using an electric field rather than chemicals, are more efficient, but they require numerous manipulations and rigorous optimizations. Additionally, electroporation methods have not yet been successfully used in large-scale preparations.
The ideal packaging system would entail using an efficient gene delivery system that is optimized for gene expression. Such a system can be based on plasmids or viral vectors (e.g., poxviruses, adenoviruses, herpesviruses, poliovirus, influenza viruses, retroviruses, etc.). Viral vectors can be used to infect a broad range of cell types in large-scale with great efficiency. Many viral vectors have been engineered for optimal gene expression and limited growth in specific cell lines.
Yet another potential limitation to using these replicon vectors is the lack of large-scale packaging systems for vector particles. The preparation of the reagents needed for packaging of, for example, alphavirus particles is costly, impractical, and not amenable to meaningful scale-up. Thus, there exists a need in the art for safe and cost-effective replicon expression vectors and packaging systems. Such vectors would be used to efficiently deliver and express psRNAV-derived RNAs for the large-scale production of infectious replicon particles for the purposes of subunit vaccine gene delivery, gene therapy, cancer immunotherapy, and recombinant protein synthesis.